Characterization of the fluorescence correlation spectroscopy (FCS) standard rhodamine 6G and calibration of its diffusion coefficient in aqueous solutions.

Characterization of the fluorescence correlation spectroscopy (FCS) standard Rhodamine 6G and calibration of its diffusion coefficient in aqueous solutions G. Majer and J. P. Melchior Citation: The Journal of Chemical Physics 140, 094201 (2014); doi: 10.1063/1.4867096 View online: http://dx.doi.org/10.1063/1.4867096 View Table of Contents: http://scitation.aip.org/content/aip/journal/jcp/140/9?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Fluorescence quenching of rhodamine-6G in Au nanocomposite polymers J. Appl. Phys. 108, 084311 (2010); 10.1063/1.3496668 Instrument response standard in time-resolved fluorescence Rev. Sci. Instrum. 80, 033109 (2009); 10.1063/1.3095677 A new high-temperature multinuclear-magnetic-resonance probe and the self-diffusion of light and heavy water in sub- and supercritical conditions J. Chem. Phys. 123, 164506 (2005); 10.1063/1.2056542 TCSPC upgrade of a confocal FCS microscope Rev. Sci. Instrum. 76, 033106 (2005); 10.1063/1.1866814 An integrated platform for surface forces measurements and fluorescence correlation spectroscopy Rev. Sci. Instrum. 74, 3067 (2003); 10.1063/1.1570947

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THE JOURNAL OF CHEMICAL PHYSICS 140, 094201 (2014)

Characterization of the fluorescence correlation spectroscopy (FCS) standard Rhodamine 6G and calibration of its diffusion coefficient in aqueous solutions G. Majer1,a) and J. P. Melchior2 1 2

Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart, Germany

(Received 6 December 2013; accepted 17 February 2014; published online 6 March 2014) Precise diffusion measurements of rhodamine 6G (Rh6G) dissolved in D2 O at concentrations between 50 and 200 μM were carried out in the temperature range from 280 to 320 K using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The obtained diffusion coefficients can be used as a calibration reference in fluorescence correlation spectroscopy (FCS). Besides measuring the diffusivity of Rh6G, the diffusion coefficient of the solvent in the same system could be determined in parallel by PFG-NMR as the resonances of water and Rh6G are well separated in the 1 H NMR spectrum. To analyze the differences due to the isotope effect of the solvent (D2 O vs. H2 O), the correlation time τ D of Rh6G was measured by FCS in both D2 O and H2 O. The obtained isotopic correction factor, τ D (D2 O)/τ D (H2 O) = 1.24, reflects the isotope effect of the solvent´s self-diffusion coefficients as determined previously by PFG-NMR. © 2014 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4867096] I. INTRODUCTION

Four decades ago, Madge, Elson, and Webb introduced fluorescence correlation spectroscopy (FCS) as a method for the measurement of the diffusion coefficients of fluorescent molecules in solutions.1 In the following years, this technique was developed further (cf., e.g., Refs. 2–4), and in 1993 the single molecule detection capability of FCS was demonstrated.5 Subsequently, FCS became widely available and found manifold applications in biology and chemistry, see, for example, Refs. 6–9, and it is also well suited for the direct study of molecular mobility in living cells.10 The principle of FCS is to measure fluorescence intensity fluctuations caused by molecules diffusing in and out of the confocal volume of a fluorescence microscope.8, 9 The autocorrelation function of the fluorescence intensity fluctuations decays with a characteristic correlation time τ D , which is closely related to the diffusion coefficient D of the diffusing molecules. However, in order to determine a diffusion coefficient from τ D , the size and the shape of the confocal volume have to be known. Since these quantities are difficult to determine independently, the diffusion coefficient of the investigated compound is usually calculated from that of a known standard by a comparison of the correlation times measured by FCS.8, 9, 11 Several authors have proposed modified concepts of FCS to circumvent these problems.12–14 These FCS variations are based on spatial cross-correlation of fluorescent signals between two volumes created by two laser foci at a known distance. However, conventional FCS can only provide absolute diffusion coefficients if the size and elongation of the confocal volume are obtained by calibration measurement of a reference compound with a known diffusion constant. a) E-mail: [email protected]. FAX: (+49) 0711 689 3612.

0021-9606/2014/140(9)/094201/6/$30.00

Because Rhodamine 6G (Rh6G) is a photostable fluorophore with a high fluorescence quantum yield, it is used extensively in biochemical applications such as fluorescence microscopy (cf., e.g., Refs. 15–18). It is also commonly used as a calibration standard in FCS measurements. Even though the diffusion coefficient of Rh6G serves as a reference to determine the dimensions of the instrumental confocal volume, literature values of D are scarce and vary considerably. A powerful tool to determine the diffusion coefficients directly, i.e., independent of any model assumptions, is pulsed field gradient nuclear magnetic resonance (PFG-NMR).19, 20 Thus, precise values of the diffusion coefficients of reference compounds for FCS measurements can be deduced from PFG-NMR data. Since NMR experiments are more easily performed in deuterated solvents, e.g., in heavy water D2 O rather than in regular water H2 O, it is necessary to correct for the isotope effect on the diffusion coefficients. This is done in the literature21 by considering the self-diffusion coefficients of the different solvents themselves, taken from published PFGNMR data,22 and assuming that the behavior of the solvents is similar to that of the solute in the solutions. When diffusion measurements had been performed at different temperatures, they were re-calculated for a reference temperature, for example 25 ◦ C, by using the well-known Stokes-Einstein equation, connecting the diffusion coefficient D with temperature T, the temperature-dependent solvent viscosity η, and the hydro-dynamic radius RH .14 These procedures introduce some uncertainty in the determination of a precise calibration standard for FCS: (i) The temperature dependence of the self-diffusion coefficient of the pure solvent, as it is deduced from the viscosity data using the Stokes-Einstein equation, is not necessarily identical to the temperature dependence of the diffusivity of the solute in the solution. (ii) The change in the self-diffusion coefficient upon fully deuterating the

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solvent may be different from the isotope effect on the diffusion coefficient of the solute dissolved in the solvent. In view of this situation, in this work we directly determined the isotope effect on the diffusion coefficient of Rh6G molecules in aqueous solutions by FCS measurements of their correlation times τ D in both H2 O and D2 O. The structure of Rh6G was characterized by measuring the 1 H NMR spectrum, which also allows inspecting the purity grade of the sample. The main aim of the present work was to precisely determine the diffusion coefficients of Rh6G in aqueous solutions at various concentrations and as a function of temperature by means of PFG-NMR. These results can be used as reference standards without the need for any corrections for the temperature variation of D based on temperature-dependent viscosities. II. EXPERIMENTAL DETAILS A. Samples

Rhodamine 6G (Rh6G, product No. 83697) was purchased from Sigma-Aldrich and the deuterated solvent D2 O (deuterium oxide, product No. HN81.3) was obtained from Carl Roth GmbH (Karlsruhe, Germany). Solutions of Rh6G in D2 O with concentrations of CRh6G = 200, 100, and 50 μM were prepared for the NMR measurements. Samples for the FCS measurements with concentrations of about CRh6G = 300 nM were prepared by dissolving very small amounts of Rh6G in either D2 O or Milli-Q water. B. FCS studies

The fluorescence correlation spectroscopy measurements were carried out at room temperature using an LSM710 confocal fluorescence microscope with a C-Apochromat 40×, 1.2 numerical aperture, water immersion objective (Zeiss, Jena, Germany). Excitation of Rh6G was performed using a cw Argon ion laser at 488 nm. Fluorescence intensity fluctuations were quantified with an avalanche photodiode detector. For both solutions, Rh6G in H2 O and in D2 O, five FCS experiments were performed, each with six replicate measurements. C. NMR spectrum

The 1 H-NMR spectrum was measured at room temperature on a Bruker Avance 300 MHz spectrometer. To improve the signal-to-noise ratio, 80 signals were added prior to the Fourier transformation. D. PFG-NMR

The pulsed field gradient NMR technique was applied to determine the diffusion coefficients D of Rh6G in aqueous solutions. The PFG measurements were performed with a Bruker Avance III 400 MHz spectrometer and a Bruker wide bore magnet. Magnetic field gradients were generated using a diff60 diffusion probe and a Great60 gradient amplifier (Bruker Biospin). All diffusivities were measured by the stimulated-echo sequence introduced by Tanner20 with a typical diffusion time of = 10 ms and an effective field-gradient

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pulse length of δ G = 1 ms. The diffusivities were determined from the dependence of the echo attenuation on the amplitude of the applied field-gradient pulses, which were varied in 16 steps between 0 and 3.5 T/m. Signal averaging was typically performed between 128 and 512 times. The diffusion probe inside the room-temperature bore of the superconducting magnet was cooled with water at a constant temperature of 25 ◦ C. Owing to the good thermal contact, this results in a sample temperature of 25 ◦ C with an accuracy of ±0.2 ◦ C without the need for any further temperature controller. PFG measurements at 25 ◦ C were carried out on samples with CRh6G = 200, 100 and 50 μM. The temperature dependence of the diffusion coefficients was measured on the sample with CRh6G = 200 μM between 280 and 320 K. For these measurements, the temperatures were maintained by means of a digital PID controller combined with ohmic heating. Temperatures below room temperature were achieved by cooling with cold nitrogen gas. The temperature was monitored with a thermocouple below the sample, which was calibrated to about ±0.1 K with a high-precision platinum resistor placed in the gas flow at the actual sample position. The maximum temperature drift during a measurement was ±0.2 K and the absolute value of the sample temperature could be determined to an accuracy of ±1 K. III. RESULTS AND DISCUSSION A. FCS measurements and isotope effect

Fluorescence correlation spectroscopy (FCS) analyzes the variations of the fluorescence intensity emitted by a very dilute fluorescent sample inside a small detection volume of a confocal microscope. The decay of the autocorrelation function of the fluorescence intensity fluctuations is attributed to molecules diffusing with a characteristic time constant τ D .8, 9 Besides diffusion through the detection volume, the population of a triplet state is another source of fluctuations in the fluorescence intensity.23 In this case, the autocorrelation function can be modeled as8, 11 −1/2 t −1 1 t 1+ G(t) = · 1+ 2 N τD S τD T t . (1) · 1+ exp − (1 − T ) τT N is the average number of fluorescent particles in the detection volume, t is the lag time, and τ D is the diffusion time. T denotes the fraction of fluorophores in the triplet state within the detection volume and τ T is the lifetime of the triplet state. The effective detection volume V = π 3/2 r3 S is characterized by its lateral size r, and its aspect ratio S. In order to calculate a diffusion coefficient D = r2 /4τ D from τ D , these quantities have to be known. Since they are difficult to determine independently, the diffusion coefficient of the investigated compound is usually related to that of a calibration standard by a comparison of the correlation times τ D measured by FCS.11 It is important to note that by measuring the diffusion time with the same confocal microscope under identical experimental conditions the ratio of the diffusion coefficients in two samples, D2 /D1 = τ D1 /τ D2 , can be determined


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FIG. 1. Fluorescence autocorrelation functions G(τ D ) of Rh6G in aqueous solutions measured by FCS at room temperature. The experimental data represent the average of five data sets with six replicate measurements each. The fitted lines correspond to correlation times of τ D = 39.0 μs for Rh6G in D2 O, and τ D = 31.4 μs for Rh6G in H2 O.

independently of size and shape of the confocal volume. In order to determine the isotope effect on the diffusion coefficients of Rh6G in aqueous solutions, we performed FCS measurements in both D2 O and H2 O. The obtained fluorescence autocorrelation functions G(τ D ) are shown in Fig. 1. The solid lines correspond to fits of Eq. (1) to the experimental data. The obtained fit parameters are τ D = 31.4 μs for Rh6G in H2 O and τ D = 39.0 μs for Rh6G in D2 O. In both cases, the fraction of triplet states was T = 0.3 and the triplet lifetime τ T = 2 μs. The ratio of the τ D values results in a ratio of the diffusion coefficients of DRh6G (H2 O)/DRh6G (D2 O) = 1.24. Within the experimental uncertainty, this result reflects the differences in the self-diffusion coefficients of the different solvents H2 O and D2 O, which were previously determined by precise PFG-NMR studies.22 B. 1 H NMR spectrum and molecular structure of Rh6G

To further characterize the Rh6G solution and to analyze its purity, 1 H NMR measurements were performed. The room-temperature 1 H NMR spectrum of Rh6G in D2 O is shown in Fig. 2. To the best of our knowledge, the 1 H NMR spectrum of Rh6G in aqueous solution has not been published so far. The pronounced peak at a chemical shift of δ = 4.8 ppm is due to remaining protons in the deuterated solvent. All other 1 H resonances can be assigned to proton positions of the Rh6G structure, as displayed in Fig. 3. No indications of any significant traces of impurities were observed. The aliphatic regions of the Rh6G molecule (see Fig. 3, positions 1–5) give rise to chemical shifts in the range of δ = 0.7–3.9 ppm. The protons of the CH3 group at position 1 yield a triplet at δ = 0.7 ppm, and those of the two CH3 groups at positions 2 result in a triplet at δ = 1.3 ppm with double intensity. The singlet at δ = 2.1 ppm is due to the CH3 groups at positions 3. The quartet assigned to the CH2 groups at positions 4 (δ = 3.4 ppm) has again twice the intensity of that assigned to the CH2 group at position 5 (δ = 3.9 ppm). The resonances

J. Chem. Phys. 140, 094201 (2014)

FIG. 2. 1 H NMR spectrum of 200 μM Rh6G in D2 O measured at room temperature. The resonance assignments of the spectrum correspond to the designations on the structure shown in Fig. 3.

of the aromatic regions (see Fig. 2, positions 6–11) occur at δ = 6.7–8.3 ppm. The protons at positions 6 and 7 give rise to singlet lines at δ = 6.7 ppm and δ = 6.9 ppm, respectively. The resonances of the protons at positions 8 and 11 occur at δ = 7.6 ppm and δ = 8.3 ppm, and both are split into doublets. The two quartets at δ = 7.9 ppm and δ = 8.0 ppm are assigned to the protons at positions 9 and 10. C. PFG-NMR measurements of the diffusivity

The diffusion coefficients D of Rh6G dissolved in D2 O were measured by pulsed field gradient nuclear magnetic resonance (PFG-NMR). In this technique, the diffusivities are deduced from the decrease in integrated intensity of a resonance peak in the NMR spectrum, δG 2 2 2 , (2) I (G) = I (0) · exp −γ G D δG − 3 with the applied field gradient G. The length of the gradient pulses δ G and the time between the two gradient pulses of a pulse sequence are kept constant. γ denotes the gyromagnetic ratio of the protons. The most convenient resonance

FIG. 3. Molecular structure and numbering for rhodamine 6G (Rh6G).


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J. Chem. Phys. 140, 094201 (2014) TABLE I. Summary of literature values of diffusion coefficients of rhodamine 6G in aqueous solution. D (10−10 m2 /s) C (μM) 3.72 ± 0.03 3.91 ± 0.06 4.02 ± 0.12 2.8 ± 0.7 ∼3 4.14 ± 0.01 4.0 ± 0.3 4.26 ± 0.13 4.14 ± 0.05

FIG. 4. Variation of the NMR signal of Rh6G with the amplitude of the applied field-gradient pulses G in a PFG-NMR measurement. The data were taken at 25 ◦ C on a sample with 200 μM Rh6G dissolved in D2 O. The solid line corresponds to a diffusion coefficient of D = 3.00 × 10−10 m2 /s.

in the 1 H NMR spectrum of Rh6G to be analyzed in the PFG-NMR experiments was the singlet at δ = 2.1 ppm (cf. Fig. 2). An example of the variation of the signal intensity with the amplitude of the applied field-gradient pulses is shown in Fig. 4. This measurement was performed on a sample with an Rh6G concentration of CRh6G = 200 μM in D2 O at a temperature of 25 ◦ C (298.15 K). The slope of the fitting curve yields a diffusion coefficient of D = 3.00 × 10−10 m2 /s. The accuracy of the least-squares fit was estimated to be ±0.5%. The analysis of the diffusion-induced attenuation of other peaks in the 1 H NMR spectrum of Rh6G yielded (except for the water peak) essentially the same diffusion coefficients. Estimating the experimental uncertainty from the standard deviation yields D = (3.00 ± 0.02) × 10−10 m2 /s for the diffusion coefficient of Rh6G in D2 O at CRh6G = 200 μM. With the isotope effect obtained by FCS, this result corresponds to a diffusion coefficient of Rh6G at a concentration of 200 μM in H2 O of D = (3.72 ± 0.03) × 10−10 m2 /s. It has been reported that Rh6G molecules have a tendency to dimerize,24, 25 which could result in a reduction in the average diffusion coefficient at high concentrations. Most of the diffusion measurements of the present work were performed on a sample with CRh6G = 200 μM. In order to study whether the average diffusion coefficient of Rh6G is higher in a sample with a lower concentration, we performed PFGNMR measurements on Rh6G at CRh6G = 100 μM in D2 O. This sample was obtained by diluting the previously studied sample by a factor of two with D2 O. Indeed, for this sample a diffusion coefficient of D = (3.15 ± 0.04) × 10−10 m2 /s was obtained, which is about 5% higher than that of the sample with CRh6G = 200 μM. After correcting for the isotope effect, this result yields for the diffusion coefficient of Rh6G in H2 O with CRh6G = 100 μM a value of D = (3.91 ± 0.06) × 10−10 m2 /s. Further diluting the sample by a factor of two again gives rise to a small but observable increase in D of about 3%. The obtained diffusion coefficients of Rh6G with

200 100 50

Technique PFG-NMR PFG-NMR PFG-NMR FCS FCS Capillary flow PFG-NMR Scanning FCS Dual-focus FCS

Temperature (◦ C) 25 25 25 22 20 25 22.5 22.5 25

Reference This work This work This work Madge2 Rigler4 Culbertson26 Gendron21 Petrasek27 Müller14

CRh6G = 50 μM in D2 O and H2 O are D = (3.24 ± 0.08) × 10−10 m2 /s and D = (4.02 ± 0.12) × 10−10 m2 /s, respectively. The results of the present work are within the range of published diffusion coefficients of Rh6G in H2 O measured at the same or at similar temperatures.2, 4, 14, 21, 26, 27 (cf. Table I). At even lower Rh6G concentrations, the diffusivity of Rh6G could no longer be measured with reasonable accuracy using PFG-NMR. This was not just prevented by the decrease in signal-to-noise ratio, which can at least partly be compensated for by increasing the number of signals to be added, but by the dominance of the water signal in the NMR spectrum. Further PFG-NMR experiments with solvent suppression are in preparation. It is interesting to note that Müller and co-workers measured the diffusion coefficient of Rh6G in H2 O at a concentration close to the infinite dilution limit in a reference-free manner by dual-focus FCS.14 They found at 25 ◦ C a value of D = (4.14 ± 0.05) × 10−10 m2 /s for the diffusivity of Rh6G. In another measurement the authors found a lower value of D = (3.89 ± 0.3) × 10−10 m2 /s, which they ascribed to the lower purity of that sample.14 Within the experimental uncertainty, the result of the present work, D = (4.02 ± 0.12) × 10−10 m2 /s for the Rh6G diffusivity in H2 O with CRh6G = 50 μM is in agreement with the D value which has been found previously for a very diluted system by dual-focus FCS.14 Another main aim of the present work was to precisely determine the temperature dependence of the diffusion coefficients of Rh6G. We performed PFG-NMR measurements at ten temperatures between about 280 and 320 K on a sample with CRh6G = 200 μM in D2 O. An Arrhenius plot of the measured diffusion coefficients of Rh6G is shown in Fig. 5 (squares). Within the investigated temperature range, the D values are well represented by a single Arrhenius law, Ha , (3) D = D0 · exp − kB T with the Boltzmann constant kB , an activation enthalpy of Ha = (230 ± 3) meV and a pre-exponential factor of D0 = (2.18 ± 0.05) × 10−6 m2 /s. It is interesting to compare these results with the self-diffusion coefficients of H2 O and D2 O, which have been measured previously by PFG-NMR over a fairly wide temperature range by Hardy et al.22 The temperature dependence of the water diffusivity cannot be


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FIG. 5. Temperature dependence of the diffusivities of Rh6G (squares) and water (circles) measured by PFG-NMR in parallel on the same sample (200 μM Rh6G in D2 O). The lines are obtained by fitting Arrhenius expressions to the diffusion coefficients. The fit parameters for Rh6G are an activation enthalpy of Ha = 0.230 eV and a pre-exponential factor of D0 = 2.18 × 10−6 m2 /s. The corresponding values for water are Ha = 0.202 eV and D0 = 4.70 × 10−6 m2 /s.

fitted to an Arrhenius law over the entire temperature range. A good description of the self-diffusion coefficients of water is obtained by a fractional power law.22 However, for the temperature range covered in the present work, the self-diffusion coefficients of H2 O and D2 O are also well represented by an Arrhenius law. The corresponding activation enthalpies are for both isotopes about Ha = 190 meV, and thus substantially smaller than the value obtained for the Rh6G diffusion. Further work will access the significance, if any, of concentration dependence on temperature dependence of the diffusion coefficients of Rh6G. A frequently used approach to analyze the temperature dependence of the Rh6G diffusion (see, e.g., Ref. 14) is to apply the Stokes-Einstein equation, kB T , D= 6π ηRh

(4)

connecting the diffusion coefficient D with the absolute temperature T, the solvent viscosity η, and the hydrodynamic radius Rh . With the temperature-dependent water viscosities from literature28 this procedure yields self-diffusion coefficients of water, which are in good agreement with the PFG-NMR results.22 In the temperature range of the present work, these data are again well described by an Arrhenius expression with an activation enthalpy of Ha = 190 meV. Thus, the present results clearly show that the temperature dependence of the Rh6G diffusion in aqueous solution (Ha = 230 meV) is stronger than that of the water self-diffusivity (Ha = 190 meV). This introduces a source of error if a diffusion coefficient of Rh6G in water measured at a given temperature is re-calculated for a different temperature only by using the temperature dependence the water selfdiffusivity. At about 25 ◦ C, a temperature change of ±1 ◦ C changes the diffusion coefficient of Rh6G by about ±3%. The

variation in the literature values (cf. Table I) may, in part, be ascribed to a lack of control over temperature, to different purity grades of the samples, or to an uncertainty in the calibration of the FCS measurements. Besides measuring the diffusivity of Rh6G, the diffusion coefficient of water in the same system was determined in parallel by analyzing the attenuation of the integrated intensity of the water peak (at δ = 4.8 ppm, cf. Fig. 2) with the applied field gradients. Owing to the better signal-to-noise ratio, the water diffusivity could be determined even more precisely than the diffusion coefficient of Rh6G. The value obtained at 25 ◦ C, Dwater (25 ◦ C) = (1.862 ± 0.005) × 10−9 m2 /s, is only slightly lower than the diffusion coefficient of traces of H2 O in pure D2 O, D = 1.902 × 10−9 m2 /s, as measured previously by Holz and Weingärtner29 at 25 ◦ C using PFG-NMR. This small reduction in Dwater is most likely due to the interaction of the water molecules with Rh6G. We also determined the temperature dependence of the water diffusivity in the same sample by PFG-NMR. For comparison with the Rh6G data, the results are included in Fig. 5 (circles). As is evident from Fig. 5, in the temperature range covered in the present work, the water diffusivity is again well described by a single Arrhenius law. The corresponding diffusion parameters are an activation enthalpy of Ha = (202 ± 3) meV and a pre-exponential factor of D0 = (4.7 ± 0.1) × 10−6 m2 /s. A comparison of these data with the temperature dependence of the self-diffusion coefficients of water indicates that the presence of Rh6G does not just slightly reduce the diffusion coefficient at room temperature but also increases the average activation enthalpy for the water diffusion. Nevertheless, Ha = 202 meV is still smaller than the activation enthalpy for the diffusion of the Rh6G molecules. This result shows again that re-calculating the diffusion coefficients of Rh6G in aqueous solutions for different temperatures on the basis of the temperature-dependent water selfdiffusivity yields inaccurate values. IV. CONCLUSION

This paper reports on a thorough characterization of the FCS standard Rh6G in aqueous solutions. Both the structure and the purity of Rh6G were studied by measuring the 1 H NMR spectrum. A main result of the present work was a precise determination of the diffusion coefficients of Rh6G and water in aqueous solutions as well as their temperature dependences by means of PFG-NMR. The diffusion coefficients of Rh6G can be used as reference standards without the need for any corrections of the temperature variation of D based on temperature-dependent viscosities. Furthermore, the isotope effect of the solvent on the diffusion coefficient is determined by FCS measurements on Rh6G dissolved in both H2 O and D2 O. ACKNOWLEDGMENTS

The authors thank Andreas Wohlfarth for valuable discussions of the 1 H NMR spectrum of rhodamine 6G, and Richard Segar for proofreading the paper. They are also grateful to Stephanie Bleicken for her help during the FCS


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measurements performed in the group of Ana J. García-Sáez at the BIOQUANT in Heidelberg. 1 D. Magde, W. W. Webb, and E. Elson, Phys. Rev. Lett. 29, 705–708 (1972). 2 D.

Magde, E. L. Elson, and W. W. Webb, Biopolymers 13, 29–61 (1974). R. Aragon and R. Pecora, Biopolymers 14, 119–137 (1975). 4 R. Rigler, P. Grasselli, and M. Ehrenberg, Phys. Scr. 19, 486–490 (1979). 5 R. Rigler, U. Mets, J. Widengren, and P. Kask, Eur. Biophys. J. Biophys. 22, 169–175 (1993). 6 Fluorescence Correlation Spectroscopy, edited by R. Rigler and E. Elson (Springer, Berlin, 2001). 7 J. Widengren and Ü. Mets in Single-Molecule Detection in Solids - Methods and Applications, edited by C. Zander, J. Enderlein, and R. A. Keller (Wiley VCH, Weinheim, 2002), pp. 69–95. 8 P. Schwille and J. Ries, Nato Sci. Peace, Sec. B 2011, 63–85. 9 J. Ries and P. Schwille, Bioessays 34, 361–368 (2012). 10 K. Bacia, S. A. Kim, and P. Schwille, Nat. Methods 3, 83–89 (2006). 11 A. J. Garcia-Saez, D. C. Carrer, and P. Schwille, Methods in Molecular Biology 606, 493–508 (2010). 12 P. S. Dittrich and P. Schwille, Anal. Chem. 74, 4472–4479 (2002). 13 T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, ChemPhysChem 8, 433–443 (2007). 14 C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, EPL 83, 46001 (2008). 3 S.

J. Chem. Phys. 140, 094201 (2014) 15 C.

Ghatak, V. G. Rao, R. Pramanik, S. Sarkar, and N. Sarkar, Phys. Chem. Chem. Phys. 13, 3711–3720 (2011). 16 J. Veilleux and S. Coulombe, Int. J. Heat Mass Transfer 53, 5321–5329 (2010). 17 I. De Santo, F. Causa, and P. A. Netti, Anal. Chem. 82, 997–1005 (2010). 18 Y. Takahashi, R. Sawada, K. Ishibashi, S. Mikuni, and M. Kinjo, Curr. Pharm. Biotechnol. 6, 159–165 (2005). 19 E. O. Stejskal and J. E. Tanner, J. Chem. Phys. 42, 288–292 (1965). 20 J. E. Tanner, J. Chem. Phys. 52, 2523–2526 (1970). 21 P. O. Gendron, F. Avaltroni, and K. J. Wilkinson, J. Fluoresc. 18, 1093– 1101 (2008). 22 E. H. Hardy, A. Zygar, M. D. Zeidler, M. Holz, and F. D. Sacher, J. Chem. Phys. 114, 3174–3181 (2001). 23 J. Widengren, U. Mets, and R. Rigler, J. Phys. Chem. 99, 13368–13379 (1995). 24 P. Ilich, P. K. Mishra, S. Macura, and T. P. Burghardt, Spectrochim. Acta, Part A 52, 1323–1330 (1996). 25 S. Dare-Doyen, D. Doizi, P. Guilbaud, F. Djedaini-Pilard, B. Perly, and P. Millie, J. Phys. Chem. B 107, 13803–13812 (2003). 26 C. T. Culbertson, S. C. Jacobson, and J. M. Ramsey, Talanta 56, 365–373 (2002). 27 Z. Petrasek and P. Schwille, Biophys. J. 94, 1437–1448 (2008). 28 W. M. Haynes, CRC Handbook of Chemistry and Physics, 93rd ed. (CRC, 2012). 29 M. Holz and H. Weingartner, J. Magn. Reson. 92, 115–125 (1991).


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